Many biological processes are mediated by the specific interaction of one protein with another. For example, enzymes are proteins that specifically bind their substrates, and substantial information is transmitted from cell to cell when ligands (such as neurotransmitters and hormones) bind their cognate receptors. Among the most fascinating interactions are those that occur in the context of an immune response in which antibodies (also known as immunoglobulins) are produced to defend the body against foreign substances that can cause infection or disease. Antibodies contain distinct domains that specifically interact with antigens and with receptors on “effector” cells, such as phagocytes. While binding the antigen is useful (in that it can prevent the antigen from interacting with its endogenous target), the most effective immune responses destroy antigens. Thus, the most effective antibodies are those with a domain that mediates high affinity antigen-binding and a domain that mediates efficient effector functions.
Naturally occurring antibodies are usually heterotetrameric; they contain two identical light (L) chains and two identical heavy (H) chains, linked together by disulfide bonds. Each heavy chain has a variable domain (VH) followed by a number of constant domains (CH1, CH2, and CH3), while each light chain has a variable domain (VL) followed by a single constant domain. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and VL is aligned with VH. The variable domains are so named because certain amino acids within them differ extensively among antibodies. These variable regions, also called complementarity determining regions (CDRs) are responsible for the binding specificity of each particular antibody for its particular antigen. Each variable domain contains three CDRs separated by highly conserved regions called framework regions (FRs). The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
The constant domains are not involved directly in binding an antibody to an antigen, but mediate various effector functions based on their binding to cellular receptor or complement molecules. Depending on the amino acid sequence of the constant region of their heavy chains, antibodies or immunoglobulins can be assigned to different classes (A, D, E, G, and M). The most commonly used therapeutic antibodies are of the “G” class (i.e., they are IgGs). These classes can be further divided. For example, IgG antibodies are further divided into the isotypes IgG1, IgG2, IgG3, and IgG4. The crystal structure of the human IgG Fc region has been determined (Deisenhofer, Biochemistry 20:2361-2370, 1981; for an illustration of the Fc region see FIG. 1 of U.S. Pat. No. 6,242,195).
The Fc region mediates effector functions that have been divided into two categories. In the first are the functions that occur independently of antigen binding; these functions confer persistence in the circulation and the ability to be transferred across cellular barriers by transcytosis (see Ward and Ghetie, Therapeutic Immunology 2:77-94, 1995). In the second are the functions that operate after an antibody binds an antigen; these functions involve the participation of the complement cascade or Fc receptor (FcR)-bearing cells. FcRs are specialized cell surface receptors on hematopoietic cells that mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g. tumor cells) coated with the corresponding antibody. Lysis occurs via antibody dependent cell mediated cytotoxicity (ADCC; see Van de Winkel and Anderson, J. Leuk. Biol. 49:511-24, 1991). FcRs are defined by their specificity for immunoglobulin isotypes. For example, Fc receptors for IgG antibodies are referred to as FcγR.
Another Fc receptor, FcRn, regulates the serum half-lives of IgGs. Enhancement or diminishment of the half-life of the Fc (or Fc-containing polypeptide) is reflected, respectively, in the increase or decrease of the Fc region affinity for FcRn (neonatal Fc receptor) (Ghetic et al., Nature Biotechnol. 15:637-640, 1997; Kim et. al., Eur. J. Immunol. 24:542-548, 1994; Della'Acqua et al. (J. Immunol. 169:5171-5180, 2002). The correlation of FcRn binding affinity and serum half-life is consistent with its proposed biological role in salvaging an antibody from lysosomal degradation. In addition, FcRn transfers IgGs from mother to fetus.
These apparently diverse roles are mediated by the ability of FcRn to transport bound IgG within and across cells. It is thought that antibodies are normally internalized from circulation by endothelial cells and are targeted to the acidic endosomes and lysosomes of the cells for degradation. FcRn is capable of binding the Fc region of an antibody at the acidic pH of an endosome (<6.5), fusing with the endothelial cell membrane, and releasing the antibody at the neutral pH of the bloodstream (>7.4), thereby salvaging the antibody from destruction. When serum antibody levels decrease, more FcRn molecules are available for IgG binding so that an increased amount of IgG is salvaged. Conversely, if serum IgG levels rise, FcRn becomes saturated, thereby increasing the proportion of antibody that is internalized and degraded (Ghetie and Ward, Annu. Rev. Immunol., (2000), 18: 739-66). Consistent with the above model, an altered polypeptide is predicted to have a longer half-life if its binding affinity for a neonatal Fc receptor is increased. Conversely, the altered polypeptide is predicted to have a shorter half-life it its binding affinity for a neonatal Fc receptor is decreased.
Monoclonal antibodies (mAbs) have now been used to treat disease in human patients (King and Adair, Curr. Opin. Drug Discovery Dev. 2:110-117, 1999; Vaswani and Hamilton, Ann. Allergy Asthma Immunol. 81:105-119, 1998; and Hollinger and Hoogenboom, Nature Biotechnol. 16:1015-1016, 1998). This is not to say that present antibody-based therapies have been entirely successful; in some instances, the limited circulation time and/or low bioavailability of a therapeutic results in a relatively low percentage of patients that exhibit a complete response to an antibody-based therapeutics, or in other cases toxicity due to prolonged circulatory half-life or exposure of non-target tissue may preclude use of the antibody as a therapy.
Accordingly, there is a need for antibodies (and other Fc-containing polypeptides such as fusion proteins) where the antigen-independent effector functions are tailored for the intended use of the antibody. Similarly, there is a need for methods that would allow for prediction of changes in antibody sequence which will alter the antigen-independent effector functions (thus obviating the need to rely on laborious trial-and-error processes). In some cases, it may be desirable to increase the half-life of the antibody. For example, protein therapeutics with an increased half-life in the blood have the advantage of concurrently decreasing the periodic dosing of the drug or alternatively to decrease the dose of the drug. Such antibodies also have the advantage of increased exposure to a disease site, e.g. a tumor. Conversely, protein therapeutics with a decreased half-life would be expected to have lower toxicity, while maintaining the efficacy that, is observed with a higher and less tolerable dose of the unaltered drug. Such therapeutics and methods or making them would be of great benefit.